IN SITU MOBILIZATION OF 35 S-LABELLED ORGANIC SULPHUR IN LITTER AND SOIL FROM A HARDWOOD FOREST

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1 Soil Biol. Biochem. Vol. 18, No. 5, pp , 1986 Printed in Great Britain /86 $3. +. Pergamon Journals Ltd IN SITU MOBILIZATION OF 35 S-LABELLED ORGANIC SULPHUR IN LITTER AND SOIL FROM A HARDWOOD FOREST T. C. STRICKLAND* and J. W. FITZGERALD Department of Microbiology, University of Georgia, Athens, GA 362, U.S.A. and W. T. SWANK Southeastern Forest Experiment Station, Coweeta Hydrologic Laboratory, Otto, NC 28763, U.S.A. (Accepted 1 March 1986) Summary 3S S-labelled inorganic sulphate (SOJ~) was incorporated into the organic matter of an extract from 2 horizon litter to yield an organic-s preparation of high specific radioactivity (459Cimmol~'; ISSOTBqmmor 1 )- In the labelled organic-s preparation, 13, 47 and 4% of the total S was present as ester sulphate, amino acid-s and sulphonate-s, respectively. The in situ mobilization of this organic- 35 S preparation was monitored in surface horizons of a hardwood forest. Of the 35 S added as organic-s, 7 and 1% was converted to SO ~ in the 2 and Al-Ap horizons, respectively after 2 days of incubation. After 7 days, up to 28% of the added S had been mineralized to SOJ~, and 76 and 4% was metabolized to other forms of organic-s in each respective horizon indicating rapid transformation rates among organic-s pools in situ. INTRODUCTION The accumulation of inorganic sulphate (SOJ") as insoluble organic-s has been observed in many ecosystems (Freney et al., 1971; Saggar et al., 1981; Strick et al., 1982; Swank efal, 1983). Once organic- S is formed, it may be mobilized to yield soluble S available for biological uptake or leaching loss (Bettany et al., 198; Johnson et al, 1982). As originally defined (Strickland et al., 1984), the term mobilization refers to the solubilization of organic-s due to depolymerization of a larger matrix to yield smaller components which may or may not undergo S-mineralization. Monitoring specific transformation dynamics within this organic-s pool has been hampered by deficiencies in methodology. 35 S was used by Freney et al. (1975) to label organic-s fractions in soil before planting with sorghum. Under these conditions, transfer of 35 S from soil organic matter to the plants was observed. However, as with the experiments of Strickland et al. (1984), such procedures require the removal of SOJ" from samples before organic-s cycling determinations, thereby creating an artificial S deficiency. These procedures can also only provide an indirect estimate of organic-s mobilization, since there is no way to non-destructively determine the amount of organic-s available for mobilization. Strickland and Fitzgerald (1984) attempted to circumvent this problem by adding 35 S-labelled organic matter free of SOj" to samples which contained in situ amounts of unlabelled SOj". The utility of this *Present address: Department of Forest Science, Oregon State University, Corvallis, OR 97331, U.S.A. S.B.B. 18/5-A 463 method was nevertheless hampered by the low specific radioactivity of the organic-s which was available, necessitating the addition of large quantities of organic- 35 S to samples. In this paper, a new method for preparing 35 S-labelled organic-s is given which provides a substrate with high specific radioactivity. Field exposure of this preparation in surface horizons of a hardwood forest was carried out and S transformations within the organic- 35 S pool were monitored for 2 and 7 days in order to obtain direct evidence for organic-s mobilization. MATERIALS AND METHODS Field exposures were carried out during July, 1984 in a mixed, mature hardwood forest located at the Coweeta Hydrologic Laboratory, near Franklin, NC. A study site adjacent to the stream (plot 9 of a permanent transect, Swank et al., 1984) was chosen to examine mobilization of organic- 35 S prepared from 2 layer material collected from the same site. The soil at this site is a sandy loam Ashe, one of the Typic Dystrochrepts. Preparation of organic from 2 layer organic matter This procedure is summarized in Fig. 1. Litter from the 2 horizon (about 3 g wet wt) was sieved (< 1 cm) to remove large roots and then shaken for 18 h at 3 C in a.1 M Na 4 P 2 C>7-NaOH buffer which had been adjusted to ph 8. with NaH 2 PO 4 crystals (litter: buffer = 1:5). This type of extraction was shown by Fitzgerald et al. (1985) to recover organic-s with minimum rupture of organic-s linkages. The

2 464 T. C. STRICKLAND et al. SIEVED 2 LITTER SHAKE 18H IN.1 M PYROPHOSPHATE BUFFER ph 8. CENTRIFUGE 2 MINUTES EXTRACT SEDIMENT (DISCARD) FILTER EXTRACT DEBRIS (DISCARD) DIALYZE 6 DAYS IN.5 M PYROPHOSPHATE BUFFER ph 8., 2 CHANGES PER DAY EXTRACT- 3Z S 4 2 SPENT BUFFER + 32 SO LOW MW ORGANICS (DISCARD) ADD GLUCOSE AND ATP. INCUBATE 24H AT 3 C. ADD 35 SO AND REINCUBATE 24H AT 3 C. DIALYZE 48H IN.5 M PYROPHOSPHATE BUFFER ph 8., 6 CHANGES PER DAY 35 S-LABELLED ORGANIC S SPENT BUFFER + GLUCOSE + ATP (DISCARD) Fig. 1. Procedure for the preparation of organic- 35 S. mixture was centrifuged, and the supernatant was filtered (2. fim) to remove floating debris. The clear filtrate (extract) was dialyzed for 6 days at 1 C against 5 HIM Na 4 P 2 O 7 -NaOH buffer, phs.o. The buffer: extract ratio was 2:1 and the buffer was changed twice daily. This dialysis completely removed all unlabelled SO^~ from the extract while retaining organic components ;> 12, daltons in molecular weight. Stock solutions of glucose and adenosine 5'-triphosphate (ATP) were sterilized by millipore filtration (.22 urn) and added to the extract to final concentrations of 2 and 5 mm, respectively. This supplemented extract was maintained at 3 C with shaking for 24 h to stimulate microbial activity. Sterile 35 S labelled SO 4 ~ (approx. 3 x 1' Bq) was then added, and the extract was held again at 3 C for 24 h with shaking. To remove residual glucose and ATP following incubation, the extract was dialyzed as above except that the buffer was changed 12 times during 48 h. After examination by electrophoresis to ensure complete incorporation of 35 S, the preparation was frozen until needed. Field exposure and sample analysis Two samples (25 x 25 x 25 cm) of the uppermost horizons of the study site were excavated and placed into containers of the same dimensions which were perforated to ensure adequate drainage. One side of each container was hinged to allow insertion of the sample with minimal disturbance, and care was taken to maintain the integrity and orientation of the 2 litter layer and soil. Sampling in this manner included the Al (-1 cm) and the Ap (1-25 cm) components of the solum. The samples in these containers were then placed in larger individual containers (catch basins) which had been sealed to prevent water loss. In order to ensure proper drainage, the interior sample containers were placed on supports to yield a 5 cm elevation from the bottom of the catch basin. Approximately 1.5 x 1 1 Bq of 35 S labelled organic-s was added to each sample. Additions of this preparation (1ml final volume) were made in 1ml increments dropwise as evenly as possible over the surface of each sample. Each addition of the label was followed by the addition of an equal volume of deionized water. The sample containers were then left open at the study site for 2 or 7 days. There was 2.3 cm of rainfall during the initial 2 day exposure and 7.8 cm accumulated between days 2 and 7. Following field exposure, the 2 and A horizons in each sample were separated and roots were removed by hand. After sieving (< 1 cm) and thorough mixing, eight sub-samples of each horizon were extracted sequentially by the method of Fitzgerald et al. (1983). This procedure yields complete recoveries of watersoluble S, salt-extractable S, and acid-alkali extractable S (insoluble organic-s). Determination of 35 S in each fraction was by liquid scintillation counting and combined yields from the three fractions was taken as 1%. Analysis by electrophoresis The organic- 35 S starting material (1 n\) and fractions derived from sample extraction after field exposure (8 fil) were analyzed by electrophoresis. In the case of the starting material, it was essential to ensure that the preparation was free of unincorporated 35 SO 4 ~. Sample fractions were analyzed to determine (i) SO 4 ~ release indicative of mineralization and (ii) forms of S not present in the starting material indicative of organic- 35 S metabolism. Electrophoresis was for 2 h at 2 V on Whatman 3 MM paper in.1 M KH 2 PO 4 -K 2 HPO 4 buffer, ph 8..

3 In situ mobilization of organic sulphur 465 (b) MOBILITY (cm) Fig. 2. Representative scans showing the electrophoretic composition of the starting material and changes associated with field exposure, (a), organic- 35 S starting material; (b), acid hydrolysate of (a); (c), water extract of O 2 horizon after 2 days exposure; (d), acid extract of O 2 horizon. The component which migrated about 13 cm from the origin is inorganic SOJ"; other components were not identified. 35 S-labelled components were located on dried strips by scanning. Components represented by peaks on the chart paper (see Fig. 2) were quantified by triangulation. Characterization of organic S pools The S status of samples was estimated for each horizon before and after field exposure. Total S was determined by the alkaline oxidation method of Tabatabai and Bremner (197), total hydriodic acid reducible-s (HI reducible-s) by the method of Johnson and Nishita (1952) as modified by Landers et al. (1983), and total non-hydriodic acid reducible-s (non-hi reducible-s) was calculated as the difference between total-s and HI reducible-s. Hydriodic acid reduction converts S in 84" and ester sulphate to sulphide which can be trapped and determined colorimetrically. Forms of S not reduced by hydriodic acid include amino acid and sulphonate-s. Raney nickel reducible-s (amino acid-s) was determined by the method of Freney et al. (197), and sulphonate-s was estimated as the difference between non-hi reducible-s and Raney nickel reducible-s. To determine phosphate extractable forms of S, sub-samples of each horizon (4 g wet wt) were mixed with.5m Na 2 HPO 4 (w/v ratio =1:5), shaken for SOmin, and centrifuged to remove particulates. All samples were treated 3 times successively in this manner and supernatant fluids were combined and filtered (2. /im retention). After determination of the HI reducible S present in each filtrate, sub-samples were filtered again successively through.45 and.22 fim membranes and the SOj~ content of each was determined by anion chromatography. Phosphate-extractable ester sulphate is the difference between the amounts of HI reducible-s and SOj"" present in each extract. The ester sulphate content remaining after this phosphate extraction is the difference between total HI reducible-s and HI reducible-s present in the phosphate extract. Table 1. Sulphur status (^gg~') of the 2 and A horizons before and after in situ exposure with 35 S-labelled organic-s' Sulphur pool Total S Total HI reducible-s Non-HI reducible-s Amino acid-s Sulphonate-S Phosphate extractable HI reducible-s Sulphate Ester sulphate Non-phosphate extractable Ester sulphate Before exposure 2-day exposure 7-day exposure 2 A.-AP 2 A r Ap 2 A,-Ap 'Means of triplicate determinations are reported

4 466 T. C. STRICKLAND et al. Table 2. Changes in sulphur composition at various stages during preparation of "S labelled organic-s from 2 layer material Stage of preparation Starting material Undialyzed extract Dialyzed extract 35 S addition and dialysis Total S <gg-') "Inorganic sulphate and ester sulphate. b Ester sulphate only. Forms of S (% Hl-reducible-S 32' 53" 41" 38" of total S available at Amino acid-s each stage) Sulphonate-S RESULTS AND DISCUSSION Characterization of samples before and after field exposure Carbon bonded-s (non-hi reducible-s) was the dominant form of S in the 2 horizon and sulphonate-s (as opposed to amino acid-s) was the major constituent of this pool (Table 1). Ester sulphate accounted for between 52 and 63% of the total S in the A horizons, while S in this linkage comprised only 25% of the S in the 2 horizon. A small proportion of S was in soluble forms as either SOj" or ester sulphate, but the amounts of these were never greater than 7 or 17% in the 2 and A horizons, respectively (see phosphate extractable-s, Table 1). Analysis of samples collected before the addition of organic-s indicated an average total S content of 98 and 218/jgSg" 1 in the 2 and A horizons, respectively. When these values are compared to total S values of the samples after field exposure it can be seen that the addition of the organic-s extract increased the S content by 34.6% in the 2 horizon while having no effect on the S content of the A horizons. The amounts of various forms of S present in samples before exposure are also listed in Table 1. Characterization of organic-s used infield exposures Table 2 summarizes the changes in composition which occurred during preparation of 35 S-labelled organic-s. The extraction of organic matter from 2 horizon litter and subsequent treatments enriched the HI reducible-s and amino acid-s content while decreasing the amounts of sulphonate-s compared to the starting material. The total S of the final preparation was comprised of 38% ester sulphate, 21% amino acid-s and 42% sulphonate-s (Table 2). These determinations include both labelled and unlabelled forms of S. In terms of 35 S-labelled components only, 47, 4 and 13% of the added 35 S was incorporated into amino acid, sulphonate and ester sulphate linkages, respectively. Further examination of the final preparation by electrophoresis revealed the presence of two components. One component remained at the origin whereas the other migrated about 9 cm from the origin (Fig. 2a). When these components were eluted separately from the paper strips and the S content of each analyzed, it was found that about 5% of the total amino acid-s (labelled and unlabelled S), 27% of the total ester sulphate and 95% of the total sulphonate-s present in the preparation remained at origin during electrophoresis. The rurm ui urgamc- o extracted Water soluble Total extracted Metabolized 11 Mineralized" Adsorbed Total extracted Metabolized Mineralized Acid extractable Total extracted Metabolized Mineralized Alkali extractable Metabolized Table 3. In situ fate of "S labelled organic-s in forest litter and soil " ± ±.3 Amount (% of total extractable 3S S) after: 2-day exposure Ar-A, 3.8 ± " 12.4 e " day exposure A,-A P 4.1 ± ± " "Mean ± SE, n = 8. "Includes organic-s transformation either by polymerization, depolymerization, desulphation or SO," incorporation. 'Conversion of S to inorganic sulphate. 'Decrease in ester sulphate (i.e. no sulphate released during acid hydrolysis). Total amount extracted with alkali = 15.3 ± 1.4, «= 8.

5 In situ mobilization of organic sulphur 467 remainder of the S in these linkage groups was present in the mobile component (Fig. 2a). In situ transformations of organic-s Differences in the electrophoretic patterns of the starting material (Fig. 2a) and of soil or litter extracts after field exposure (Fig. 2c,d) enabled the determination of in situ transformations of organic-s (summarized in Table 3). In both horizons, much of the added 35 S became insoluble after 2 days and was thus not extractable with l.om salt solutions. The amount of salt-extractable 35 S became further diminished between days 2 and 7 in the 2 horizon while, this pool increased in the A horizons. Thus, although the organic-s was soluble in pyrophosphate buffer, its addition to field samples resulted in >7% of it being located in the acid-alkali extractable fraction, thereby indicating the insolubility of the organic-s in situ. In the A horizons, there was a decrease in acid-alkali extractable S between days 2 and 7 which resulted in an increase in soluble and adsorbed S pools. The increase in these latter fractions was manifested as an increase in SOj", while the amounts of soluble and adsorbed organic-s diminished in the A horizons. Conversely, in the 2 horizon the amounts of soluble and adsorbed organic-s, as well as SOj~, decreased during this period (Table 3). These results suggest that in the 2 horizon, organic-s accumulating and polymerizing processes dominate, resulting in the production of an insoluble organic-s matrix. While high rates of mobilization occurred early during field exposures in the A horizons, decreased amounts of soluble and adsorbed organic-s observed after 7 days suggest that these rates became diminished. When the supply of soluble organic-s becomes exhausted, the rates of S-mineralization will be governed by the rate of depolymerization (solubilization) of the insoluble organic-s matrix in situ. Net changes in organic-s metabolism (Table 4) indicate that S- mineralization was about 2-fold greater after 7-days in both horizons. This indicates that conversion of organic-s to SOj~ is a continuous process in situ even when there is an excess of SO*" from precipitation. After the 7-day exposure, about 3.5 ^g I" 1 of SOJ"-S had entered the study site in throughfall. Table 4 also shows that total organic-s'transformation (metabolism and mineralization) ranged from about 9% of added S in the 2 horizon to 8% in the A horizons after 7 days. This suggests that although there is a net retention of S in these horizons (Swank and Douglass, 1977), a large capacity for organic-s metabolism does exist. The assessment of organic-s cycling in situ has been hampered by limitations to detection and by incomplete characterization of the organic-s pool Table 4. Cycling of organic-s in forest floor and soil during 2 and 7 day field exposures Amount (%) of added organic-s: Metabolized 8 Mineralized Horizon 2-day 7-day 2-day 7-day 2 A,-Ap " See footnote b, Table 3 for explanation. b Unable to detect change in starting material (Strickland et al., 1984; Fitzgerald et al., 1985). However, the method of preparation utilized in our study provided organic-s with sufficiently high specific radioactivity to overcome limitations to detection when assaying large (kg) quantities of soil. In developing the procedure, advantage was taken'of the observation that this carbon- and energy-dependent process is microbially mediated (Fitzgerald et al., 1983). Moreover, the use of an organic matter extract as opposed to 2 layer material per se (Strickland and Fitzgerald, 1985), ensured that adsorption of added label did not take place thus enriching the radioactivity of the final preparation. Characterization of this preparation with respect to linkage types and electrophoretic patterns made it possible to monitor the rapid exchange of S between different pools. For example, organic-s mineralization was detected not only by the presence of SOj~ but also by a net increase in ester sulphate content (carbon bonded-s must be mineralized to supply SOj~ for esterification). Moreover, a net decrease in ester sulphate content is also indicative of mineralization. Further insight into the cycling of organic-s can be obtained from the appearance in extracts of electrophoretically-separable components which were not present in the starting material. Such components may be produced from the mineralization and reincorporation of S, or they may be depolymerization or polymerization products of components already present in the starting material. We propose that this approach has resulted in a more comprehensive view of organic-s mobilization. Acknowledgements This research was supported by the National Science Foundation and through a cooperative agreement with the United States Department of Agriculture (Forest Service). We thank }. T. Ash and D. D. Hale for expert technical assistance, M. E. Watwood and W. T. Scott Swank for sampling assistance, and B. F. Strickland for proofreading. REFERENCES Bettany J. R., Saggar S, and Stewart J. W. B. (198) Comparison of the amounts and forms of sulfur in soil organic matter fractions after 65 years of cultivation. Soil Science Society of America Journal 44, Fitzgerald J. W., Ash J. T., Strickland T. C. and Swank W. T. (1983) Formation of organic sulfur in forest soils: a biologically mediated process. Canadian Journal of Forest Research 13, Fitzgerald J. W., Strickland T. C. and Ash J. T. (1985) Isolation and partial characterization of recently formed forest floor and soil organic sulfur. Biogeochemistry 1, Freney J. R., Melville G. E. and Williams C. H. (197) The determination of carbon bonded sulfur in soil. Soil Science 19, Freney J. R., Melville G. E. and Williams C. H. (1971) Organic sulphur fractions labelled by the addition of 35 S-sulphate to soil. Soil Biology & Biochemistry 3, Freney J. R., Melville G. E. and Williams C. H. (1975) Soil organic matter fractions as sources of plant-available sulphur. Soil Biology & Biochemistry 7, Johnson D. W., Henderson G. S., Huff D. D., Lindberg S. E., Richter D. D., Shriner D. S., Todd D. E. and Turner J. (1982) Cycling of organic and inorganic sulphur in a chestnut oak forest. Oecologia 54,

6 468 T. C. STRICKLAND et al. Johnson C. M. and Nishita H. (1952) Microestimation of sulfur in plant materials, soil, and irrigation waters. Analytical Chemistry 24, Landers D. H., David M. D. and Mitchell M. J. (1983) Analysis of organic and inorganic sulfur constituents in sediments, soils and water. International Journal of Environmental and Analytical Chemistry 14, Saggar S., Bettany J. R. and Stewart J. W. B. (1981) Sulfur transformations in relation to carbon and nitrogen in incubated soils. Soil Biology & Biochemistry 13, Strick J. E., Schindler S. C., David M. B., Mitchell, M. J. and Nakas J. P. (1982) Importance of organic sulfur constituents and microbial activity to sulfur transformations in an Adirondack forest soil. Northeastern Environmental Science 1, Strickland T. C. and Fitzgerald J. W. (1984) Formation and mineralization of organic sulfur in forest soils. Biogeochemistry 1, Strickland T. C., Fitzgerald J. W. and Swank W. T. (1984) Mobilization of recently formed forest soil organic sulfur. Canadian Journal of Forest Research 14, Strickland T. C. and Fitzgerald J. W. (1985) Incorporation

/88 $ Copyright 1988 Pergamon Press pic

/88 $ Copyright 1988 Pergamon Press pic Soil Biol. Biochem. Vol. 20, No. 6, pp. 825-831, 1988 Printed in Great Britain. l rights reserved 0038-071788 $3.00 + 0.00 Copyright 1988 Pergamon Press pic SULPHUR-CONTAINING AMINO ACID METABOLISM IN